The present invention relates to mass spectrometry. In particular, the present invention relates to improvements in matrix assisted laser desorption ionization time of flight mass spectrometry.
With the advent of delayed extraction (DE) or time-lag energy focusing technology, matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) has enjoyed renewed interest in analyzing samples of biological origin.
In MALDI-TOF, ions of a sample to be analyzed, which are generated and desorbed by laser or other ionization source, are sensed by a detector remote from the sample. By measuring the time it takes the generated ions to travel to the detector, different ions can be separated from one another according to their mass-to-charge ratios. In DE, the sample holder on which the sample is mounted is pulsed with an electric potential at a predetermined time after ionization. As a result, an electric field is set up which extracts sample ions from the ion plume and accelerates the extracted ions towards the detector. The overall result is that accuracy and sensitivity of the analysis is enhanced. See U.S. Pat. No. 5,760,393, the disclosure of which is incorporated herein by reference.
A major advantage of MALDI-TOF is that multiple samples can be analyzed in a single run of the machine. For example, some commercially-available MALDI-TOF mass spectrometers use sample holders accommodating up to 100 different samples arranged, for example, in a 10xc3x9710 grid. The machine can be programmed to sequentially analyze each of these samples in order, thereby allowing the machine to automatically perform 100 different analyses in a single run of the machine.
A current objective of modern science is to completely sequence the human genome. An important part of this effort will be analyzing single nucleotide polymorphisms (SNP""s), the most common genetic variation in the human genome pool. Because of its sensitivity and ability of analyzing multiple samples in a high-throughput manner, MALDI-TOF appears to be one of the best technologies for this effort.
However, current MALDI-TOF technology is still limited for this purpose. For example, mass differences between nucleotides can be as little as 9 Da (the difference between the A and T heterozygotes), and current DE-modified MALDI-TOF technology cannot resolve this difference unambiguously. See Fei et al. Nucleic Acids Research, Vol. 26, Page 2827, 1998. Furthermore, multiplex genotyping of SNP""s requires the very best resolution over a broad mass range, while current DE-modified MALDI-TOF technology provides good resolution only in a narrow mass range, at least when using a single set of focusing parameters (i.e. in a single run of an automated machine). See Juhasz et al., Analytical Chemistry, Vol. 68, Page 941, 1999.
Still another disadvantage of current DE-modified MALDI-TOF technology relates to sample substrates. It has been demonstrated that in many cases, MALDI sample substrates made from dielectric materials provide better sensitivity than those made from metals. Hence, samples in MALDI-TOF analyses are often mounted on a dielectric sheet carried by the sample holder, rather than directly on the sample holder itself. To this end, it has also been proposed to directly analyze proteins separated by 2-D gels by attaching the gels or membranes directly to the MALDI sample holder. See Ogorzalek Loo et al., Electrophoresis, Vol. 18, page 382, 1997. However, such dielectric sheets also cause localized variations in electrical potential from sample to sample, as well as from spot to spot on the same sample, during machine operation. Although the addition of internal standards or reference chemicals for calibration purposes can overcome this problem to a certain degree, these variations make it difficult to generate high-quality spectra by multiple data acquisitions.
Accordingly, it is an object of the present invention to provide a new approach to MALDI-TOF mass spectrometry which provides better accuracy, sensitivity and resolution than current technology and which further allows easier data acquisition and other advantages compared with past practice.
This and other objects are accomplished by the present invention which is based on the discovery that improved MALDI-TOF mass spectrometry can be achieved by spatially separating the electric field used for extraction/acceleration from the location of the sample in the machine. In particular, it has been found that by applying the electrical potential used for extraction/acceleration to a focusing element spaced apart from the sample, rather than to the sample itself or the sample holder carrying the sample, superior accuracy and resolution can be achieved.
Accordingly, the present invention provides new technology that can substantially improve the performance of delayed extraction MALDI-TOF mass spectrometry in analyzing oligonucleotide and other biomolecules. Unlike conventional delayed extraction MALDI-TOF, this new approach separates desorption/ionization from acceleration. Hence, the acronym SDIFA. With SDIFA, isotope-limited resolution can be obtained for oligonucleotides of up to 62 mers, and multiple T/A heterozygote samples in the mass range of 5200-7800 Da can be clearly identified using a single set of focusing parameters, i.e. in a single run of the machine. Moreover, compared with conventional delayed extraction MALDI-TOF mass spectrometry, the performance of SDIFA is more stable and less dependent on the experimental conditions such as laser power, sample spots, delay times and the extraction field. The result is that data acquisition is both easier and more reproducible.
Thus, the present invention provides a new process and apparatus for carrying out MALDI-TOF mass spectrometry in which the electric field for achieving extraction of sample ions from the ion plume created by sample ionization, and acceleration of those ions towards the detector of the spectrometer, is spatially separated from the sample.
In particular the present invention provides a new process and apparatus for carrying out MALDI-TOF mass spectrometry in which the electric potential for achieving extraction and acceleration of sample ions is applied to a focusing element of the device spaced apart from the sample holder, rather than the sample holder or sample itself as done in past practice.
In accordance with a further aspect of the invention, it has also been found that the superior performance of the inventive SDIFA technology allows gel sheets and other separation (adsorption) media traditionally used for physically separating closely-related compounds to be directly used as targets in the mass spectrometric analysis. The traditional approach for analyzing multiple closely-related compounds such as occurs in protein analysis, for example, is to (1) physically separate the compounds by adsorption, (2) extract each compound individually from the adsorption medium, (3) deposit each extracted compound on a common sample holder, and (4) separately analyze each compound. Because of the superior performance of the inventive SDIFA technology, however, it has been found that high quality analytical results can be obtained by directly using the adsorption medium as the analysis target. This approach totally eliminates steps (2) and (3) in the above protocol, thereby vastly simplifying automated data acquisition.
Thus the present invention also provides an improvement in processes for automatic mass spectrometric analysis of multiple samples recovered on a common separation medium, the improvement wherein the common separation medium is directly used as the target in a mass spectrometric analysis carried out using the SDIFA technology of the present invention.